Method for improving memory of a subject using a composition comprising cistanche and ginkgo extracts

ABSTRACT

A method and composition useful for improving memory of a subject are disclosed. The method comprises the step of administering the composition to the subject. The composition consists essentially of  Cistanche  extract and  Ginkgo  extract (e.g. as actives). In a first general embodiment, the extracts are present in a weight ratio ( Cistanche  to  Ginkgo ; or “C:G”) that is &gt;2.5:1. In a second general embodiment, the extracts are present in a weight ratio that is &lt;2.5:1. In embodiments of the first general embodiment, the composition comprises about 72-99 weight percent (wt. %)  Cistanche tubulosa  extract and about 1-28 wt. %  Ginkgo biloba  extract. In embodiments of the second general embodiment, the composition comprises about 50-70 wt. %  Cistanche tubulosa  extract and about 30-50 wt. %  Ginkgo biloba  extract. The  Cistanche tubulosa  extract is generally obtained from root material and the  Ginkgo biloba  extract is generally obtained from leaf material. The composition may also include inactives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and all advantages of U.S. Pat. App.No. 61/990,200 filed on May 8, 2014 and U.S. Pat. App. No. 62/081,104filed on Nov. 18, 2014, the content of which is hereby incorporated byreference

FIELD OF THE INVENTION

The present invention generally relates to a method and composition forimproving memory of a subject. The method comprises the step ofadministering the composition to the subject. The composition consistsessentially of Cistanche and Ginkgo extracts.

DESCRIPTION OF THE RELATED ART

Short-term memory is supported by transient patterns of neuronalcommunication, dependent on regions of the frontal and parietal lobes.On the other hand, long-term memory is maintained by more stable andpermanent changes in neural connections widely spread throughout thebrain. It is believed that the hippocampus is essential to theconsolidation of information from short- to long-term memory, althoughit does not seem to store information itself.

A number of efforts have been made to improve short- and long-termmemories. For example, dietary supplements have been provided in aneffort to improve brain health and mental performance. A specificexample of such is the “Memory Builder™ with Ginkgo” dietary supplementfrom NUTRILITE® of Buena Park, Calif., US. This dietary supplement is inthe form of a tablet consisting of the following active ingredients: 300mg Cistanche tubulosa extract (root) and 120 mg Ginkgo biloba extract(leaves).

While various efforts have been made, there remains an opportunity toprovide additional methods and compositions for improving brain health,memory formation, and/or memory retention. Moreover, there remains anopportunity to reverse, slow, or prevent memory loss.

BRIEF SUMMARY OF THE INVENTION

A method and a composition are disclosed. The method and composition areuseful for improving memory of a subject. The method comprises the stepof administering the composition to the subject. The compositionconsists essentially of Cistanche extract and Ginkgo extract. In a firstgeneral embodiment, the extracts are present in a weight ratio(Cistanche to Ginkgo; or “C:G”) that is >2.5:1. In a second generalembodiment, the extracts are present in a weight ratio (C:G) that is<2.5:1.

In various embodiments of the first general embodiment of thisdisclosure, the composition comprises about 72-99 weight percent (wt. %)Cistanche tubulosa extract and about 1-28 wt. % Ginkgo biloba extract.In these embodiments, the Cistanche tubulosa and Ginkgo biloba extractsare present in a weight ratio (C:G) that is from 2.6:1 to 20:1. Invarious embodiments of the second general embodiment of this disclosure,the composition comprises about 50-70 wt. % Cistanche tubulosa extractand about 30-50 wt. % Ginkgo biloba extract. In these embodiments, theCistanche tubulosa and Ginkgo biloba extracts are present in a weightratio (C:G) that is from 1:1 to 2.4:1. The Cistanche tubulosa extract isgenerally obtained from root material and the Ginkgo biloba extract isgenerally obtained from leaf material.

Without being bound or limited by any particular theory, it is thoughtthat the method and composition of this disclosure are useful forimproving brain health, memory formation, and/or memory retention.Moreover, it is thought that the method and composition of thisdisclosure are useful for reversing, slowing, or preventing memory loss.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the disclosure will be readily appreciated, as thesame becomes better understood by reference to the following detaileddescription when considered in connection with the accompanying drawingswherein:

FIG. 1A depicts bar charts of acute application examples;

FIG. 1B depicts bar charts of further acute application examples;

FIG. 1C depicts bar charts of further acute application examples;

FIG. 1D depicts bar charts of further acute application examples;

FIG. 2 depicts bar charts of chronic application examples;

FIG. 3 depicts line graphs of chronic application examples;

FIG. 4 depicts bar charts of chronic application examples;

FIG. 5A is an image associated with semi-automatic quantification ofexamples;

FIG. 5B is an image corresponding to synapse channel for examples;

FIG. 6 illustrates examples of an acute study to assess mixtures ofCistanche and Ginkgo and effective concentration range;

FIG. 7A is a bar chart of examples of a study to assess combinatorialeffects of Cistanche and Ginkgo;

FIG. 7B is a line graph of examples further illustrating the study toassess combinatorial effects of Cistanche and Ginkgo;

FIG. 7C is a line graph of examples further illustrating the study toassess combinatorial effects of Cistanche and Ginkgo;

FIG. 8 illustrates examples of a study to assess chronic effects ofCistanche tubulosa extract on maturation of a neuronal network;

FIG. 9 illustrates examples of a study directed at immuno-histochemicalanalysis;

FIG. 10 illustrates a Cistanche tubulosa fingerprint;

FIG. 11 illustrates a Ginkgo biloba fingerprint;

FIG. 12 illustrates steps of a phenotypic screening process;

FIG. 13 depicts various neuron/glia co-cultures;

FIG. 14 illustrates multi-parametric characterization of neuronalnetwork activity;

FIG. 15 depicts line graphs of acute application of Ginkgo bilobaexamples;

FIG. 16 depicts line graphs of acute application of Cistanche tubulosaexamples;

FIG. 17 depicts Ginkgo and Cistanche mixture and concentration matrices;

FIG. 18A depicts bar charts illustrating chronic effects of examples;

FIG. 18B depicts additional bar charts illustrating chronic effects ofexamples;

FIG. 19A is a first portion of a heat map;

FIG. 19B is second portion of the heat map of FIG. 19A;

FIG. 20A is a bar chart illustrating chronic application effects onhippocampal network morphology of examples;

FIG. 20B is a bar chart illustrating chronic application effects onhippocampal network morphology of examples;

FIG. 20C is a bar chart illustrating chronic application effects onhippocampal network morphology of examples;

FIG. 20D is a bar chart illustrating chronic application effects onhippocampal network morphology of examples; and

FIG. 21 are images associated with semi-automatic quantification ofexamples.

DETAILED DESCRIPTION OF THE INVENTION

A method and a composition are disclosed. The method and composition areuseful for improving memory of a subject. The method comprises the stepof administering the composition to the subject. Without being bound orlimited by any particular theory, it is thought that the method andcomposition of this disclosure are useful for improving brain health,memory formation, and/or memory retention. Moreover, it is thought thatthe method and composition of this disclosure are useful for reversing,slowing, or preventing memory loss. Other potential non-limitingbenefits are described herein.

The composition consists essentially of Cistanche extract and Ginkgoextract. As used herein, the phrase “consisting essentially of”generally encompasses the specifically recited elements/components for aparticular embodiment. Further, the phrase “consisting essentially of”generally encompasses and allows for the presence of additional oroptional elements/components that do not materially impact the basicand/or novel characteristics of that particular embodiment. In certainembodiments, “consisting essentially of” allows for the presence of ≦10,≦5, or ≦1, weight percent (wt. %) of additional or optional componentsbased on the total weight of the composition. In other embodiments, thecomposition consists of Cistanche extract and Ginkgo extract asdescribed herein. The aforementioned extracts may be referred to hereinas the extracts, actives, or active ingredients. In various embodiments,actives of the composition consist of the Cistanche (or “HerbaCistanche”) and Ginkgo (or “Yin Xing Ye”) extracts.

Components that would generally materially impact the method/compositionof this disclosure include active ingredients that are different fromCistanche extract and Ginkgo extract. In certain embodiments, thecomposition of this disclosure is free of other active ingredients. By“other active ingredients”, it is generally meant that the compositionis free of other types of Traditional Chinese Medicines (“TCMs”; or“Chinese medicines”) that are different from Cistanche extract andGinkgo extract. Other types of TCMs are understood in the art. Examplesof other types of TCMs are generally described as “bioactive substances”in International Pub. No. WO01/22934A2, the content of which isincorporated by reference in its entirety.

In certain embodiments, the composition of this disclosure can compriseinactive ingredients as described below. If utilized, the inactiveingredients are different from Cistanche, Ginkgo, and other activeingredients.

Components that would not generally impact the method/composition ofthis disclosure include inactive ingredients. Inactive ingredients areunderstood in the art and are different from active ingredients, such asthose described above. Examples of inactive ingredients include, but arenot limited to, flavorings; carob; corn syrups, such as hydrolyzed cornsyrup solids; cellulose, such as methyl cellulose, hydroxypropyl methylcellulose, carboxy methyl cellulose, microcrystalline cellulose, andpowdered cellulose; fructose; maltodextrin and maltol, such as naturalmaltol; sorbitol; preservatives; alcohols, such as ethanol, propylalcohol and benzyl alcohol; glycerin; potassium sorbate; sodiumbenzoate; binders; flow agents; stearates, such as calcium stearate,magnesium stearate, and sodium magnesium stearate; dicalcium phosphate;glyceryl triacetate; vegetable oils, such as hydrogenated vegetableoils; mineral oils; water; silicones, such as silicone oils; silicondioxide; stearic acid; waxes, such as carnauba wax and beeswax;starches, such as corn starch and potato starch; fatty esters and fattyalcohols; glycols and polyglycols; and combinations thereof. If utilizedto form the composition, the inactive ingredient(s) can be used invarious amounts. Further, it is to be appreciated that the amounts ofactives described herein can be normalized with respect to 100 parts byweight of the composition to account for the presence of inactiveingredients (if utilized). This disclosure is not limited to aparticular inactive ingredient or amount thereof.

The composition includes Cistanche extract. The Cistanche extract isobtained from plant material from the genus Cistanche. Cistanche is aworldwide genus of holoparasitic desert plants in the order Lamiales,family Orobanchaceae. Notable species of this genus include Cistanche(C.) ambigua, C. deserticola, C. phelypaea, C. salsa, C. sinensis, andC. tubulosa. These species can be found in the arid lands of China andPakistan and other parts of the world. In certain embodiments, C.deserticola is not utilized based on scarcity.

In many embodiments, the Cistanche extract is an extract from thespecies Cistanche tubulosa (Shrenk.) Wight (also known simply asCistanche tubulosa). Cistanche (root) may also be referred to as “HerbaCistanche” according to the Chinese Pharmacopeia. Herba Cistanche mayalso be referred to as Rou Cong Rong,

,

,

, Desertliving Cistanche, Cong Rong,

,

, or

.

As used herein, reference to “Cistanche extract” generally refers to anextract containing material from the genus Cistanche including from thespecies Cistanche tubulosa. Optionally, other Cistanche species may beused in addition or alternate to Cistanche tubulosa; however, inclusionof Cistanche tubulosa extract is generally preferred. The Cistancheextract may be commercially obtained from various resources. Inaddition, suitable Cistanche extracts can be obtained by using anyconventional extraction technique including, but not limited to, one ormore techniques described further below.

Any part of the Cistanche plant may be used to obtain the extract usedin the composition including, but not limited to, the root, stem,rhizome, leaf, flower, fruit, and/or extracts of these parts. Cistancheactives are generally found in the root of Cistanche plants and as such,root extraction (or root extract) is generally most useful for purposesof this disclosure. The Cistanche may be used in raw form, suspendedform, dehydrated form, concentrated form, or extract form. In specificembodiments, the Cistanche extract is obtained from root material of aplant in the genus Cistanche, e.g. from root material of one or moreCistanche tubulosa plants.

Extracts of Cistanche roots may contain various actives (orphytochemicals), such as phenylethanoid glycosides. Without being boundor limited by any particular theory, it is thought that activecomponents of Cistanche have positive effects on neuronal health,specifically as an anti-oxidant neuroprotective and as anendothelium-dependent relaxant to help promote optimal blood flow to thebrain. Further, the mechanisms by which Cistanche supports neuronalstrength are posited to be via increasing neuronal growth factors andinhibiting neurotransmitter breakdown.

In various embodiments, the Cistanche extract comprises at least onephenylethanoid glycoside, or mixtures thereof, in an amount of at leastabout 10, at least about 20, at least about 30, at least about 40, atleast about 50, at least about 60, or at least about 70, %. Moreover,the Cistanche extract comprises at least one acteoside, cistanoside,echinacoside, or isoacteoside, or mixtures thereof, in an amount of atleast about 1, at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 15, at least about 20,at least about 25, at least about 30, at least about 35, or at leastabout 40, %. In all of these embodiments, an upper boundary is 100%. Itis contemplated that any and all values or ranges of values betweenthose described above may also be utilized.

The composition also includes Ginkgo extract (which may also be referredto as “Gingko extract” or “maidenhair tree” extract). The Ginkgo extractis obtained from plant (or tree) material from the genus Ginkgo. Ginkgois a worldwide genus of plants in the order Ginkgoales, familyGinkgoaceae. Notable species of this genus include Ginkgo (G.)aditantoides, G. apodes, G. biloba, G. cranei, G. digitata, G. dissecta,G. gardneri, G. ginkgoidea, G. huolinhensis, G. huttonii, and G.yimaensis. These species can be found in China and other parts of theworld.

In many embodiments, the Ginkgo extract is an extract from the speciesGinkgo biloba. Ginkgo (leaf) may also be referred to as “Yin Xing Ye”according to the Chinese Pharmacopeia. Yin Xing Ye is also referred toas

, Ginkgo Leaf,

,

,

, or Folium Ginkgo. Ginkgo (nut) may also be referred to as Bai Guo,although Yin Xing Ye is generally preferred over Bai Guo.

As used herein, reference to “Ginkgo extract” generally refers to anextract containing material from the genus Ginkgo including from thespecies Ginkgo biloba. Optionally, other Ginkgo species may be used inaddition or alternate to Ginkgo biloba; however, inclusion of Ginkgobiloba extract is generally preferred. The Ginkgo extract may becommercially obtained from various resources. In addition, suitableGinkgo extracts can be obtained by using any conventional extractiontechnique including, but not limited to, one or more techniquesdescribed further below.

Any part of the Ginkgo plant may be used to obtain the extract used inthe composition including, but not limited to, the root, stem, rhizome,leaf, flower, fruit, and/or extracts of these parts. Ginkgo actives aregenerally found in the leaves of Ginkgo plants and as such, leafextraction (or leaf extract) is generally most useful for purposes ofthis disclosure. The Ginkgo may be used in raw form, suspended form,dehydrated form, concentrated form, or extract form. In specificembodiments, the Ginkgo extract is obtained from leaf material of aplant in the genus Ginkgo, e.g. from leaf material of one or more Ginkgobiloba plants (i.e., Ginkgo biloba L.).

Extracts of Ginkgo leaves may contain various actives (orphytochemicals), such as flavonoid glycosides (e.g. myricetin andquercetin) and terpenoids (e.g. ginkgolides and bilobalides). Ginkgoplant material may also contain terpene trilactones (e.g. ginkgolides A,B, C, J and bilobalide), flavonol glycosides, biflavones,proanthocyanidins, alkylphenols, simple phenolic acids,6-hydroxykynurenic acid, 4-O-methylpyridoxine, and polyprenols. Withoutbeing bound or limited by any particular theory, it is thought thatactive components of Ginkgo improve blood flow to the brain and act asantioxidants. Further, it is thought that memory and cognitive speed mayalso be improved.

In various embodiments, the Ginkgo extract comprises at least oneflavonoid, or mixtures thereof, in an amount of at least about 1, atleast about 5, at least about 10, at least about 15, at least about 20,at least about 22, at least about 24, at least about 25, at least about30, at least about 35, or at least about 40, %. Moreover, the Ginkgoextract comprises at least one terpenoid, or mixtures thereof, in anamount of at least about 0.1, at least about 0.5, at least about 1, atleast about 2, at least about 3, at least about 4, at least about 5, atleast about 6, at least about 7, at least about 8, at least about 9, atleast about 10, at least about 15, or at least about 20, %. In all ofthese embodiments, an upper boundary is 100%. It is contemplated thatany and all values or ranges of values between those described above mayalso be utilized.

In a first general embodiment of this disclosure (“first embodiment”),the extracts are present in a weight ratio (Cistanche to Ginkgo; or“C:G”) that is >2.5:1. In other words, the Cistanche extract is presentin an amount more than 2.5 times that of the Ginkgo extract. Typically,the weight ratio is from 2.6:1 to 20:1, 2.8:1 to 15:1, 3:1 to 9:1, 3:1to 8:1, 3:1 to 7:1, 3:1 to 6:1, 3:1 to 5:1, 3:1 to 4:1, or 3:1.Alternatively, the weight ratio is from 2.6:1 to 20:1, 2.8:1 to 15:1,3:1 to 9:1, 4:1 to 9:1, 5:1 to 9:1, 6:1 to 9:1, 7:1 to 9:1, 8:1 to 9:1,or 9:1. It is contemplated that any and all values or ranges of valuesbetween those described above may also be utilized.

In various embodiments of the first embodiment, the Cistanche extract ispresent in an amount of from about 72-99, about 73-98, about 74-97,about 75-96, about 75-95, or about 75-90, wt. % based on 100 parts byweight of the composition. In specific embodiments, the Cistancheextract is present in an amount of from about 72-99, about 75-98, about80-97, about 85-96, about 90-95, or about 90, wt. % based on 100 partsby weight of the composition. In other specific embodiments, theCistanche extract is present in an amount of from about 72-99, about73-95, about 74-90, about 75-85, about 75-80, or about 75, wt. % basedon 100 parts by weight of the composition. It is contemplated that anyand all values or ranges of values between those described above mayalso be utilized.

Moreover, the Ginkgo extract is present in an amount of from about 1-28,about 2-27, about 3-28, about 4-27, about 5-26, about 6-25, about 7-25,about 8-25, about 9-25, or about 10-25, wt. % based on 100 parts byweight of the composition. In specific embodiments, the Ginkgo extractis present in an amount of from about 1-28, about 2-25, about 3-20,about 4-15, about 5-10, or about 10, wt. % based on 100 parts by weightof the composition. In other specific embodiments, the Ginkgo extract ispresent in an amount of from about 1-28, about 5-27, about 10-26, about15-25, about 20-25, or about 25, wt. % based on 100 parts by weight ofthe composition. It is contemplated that any and all values or ranges ofvalues between those described above may also be utilized.

In certain embodiments of the first embodiment, the compositioncomprises about 72-99 wt. % Cistanche tubulosa extract and about 1-28wt. % Ginkgo biloba extract. The Cistanche tubulosa and Ginkgo bilobaextracts are present in a weight ratio (C:G) that is from 2.6:1 to 20:1.Moreover, the Cistanche tubulosa extract is obtained from root materialand the Ginkgo biloba extract is obtained from leaf material. Inspecific embodiments, the Cistanche tubulosa extract is present in anamount of about 90 wt. % and the Ginkgo biloba extract is present in anamount of about 10 wt. %, each based on 100 parts by weight of thecomposition. In other specific embodiments, the Cistanche tubulosaextract is present in an amount of about 75 wt. % and the Ginkgo bilobaextract is present in an amount of about 25 wt. %, each based on 100parts by weight of the composition. It is contemplated that any and allvalues or ranges of values between those described above may also beutilized.

In a second general embodiment of this disclosure (“second embodiment”),the extracts are present in a weight ratio (C:G) that is <2.5:1. Inother words, the Cistanche extract is present in an amount less than 2.5times that of the Ginkgo extract. Typically, the Cistanche extract ispresent in an amount equal to or greater than, more typically greaterthan, that of the Ginkgo extract. Typically, the weight ratio is from1:1 to 2.4:1, 3:2 to 7:3, 2:1 to 7:3, 2.1:1 to 7:3, 2.2:1 to 7:3; 2.3:1to 7:3; or 7:3. It is contemplated that any and all values or ranges ofvalues between those described above may also be utilized.

In various embodiments of the second embodiment, the Cistanche extractis present in an amount of from about 50-70, about 55-70, about 60-70,about 65-70, or about 70, wt. % based on 100 parts by weight of thecomposition. It is contemplated that any and all values or ranges ofvalues between those described above may also be utilized.

Moreover, the Ginkgo extract is present in an amount of from about30-50, about 30-45, about 30-40, about 30-40, about 30-35, or about 30,wt. % based on 100 parts by weight of the composition. It iscontemplated that any and all values or ranges of values between thosedescribed above may also be utilized.

In certain embodiments of the second embodiment, the compositioncomprises about 50-70 wt. % Cistanche tubulosa extract and about 30-50wt. % Ginkgo biloba extract. The Cistanche tubulosa and Ginkgo bilobaextracts are present in a weight ratio (C:G) that is from 1:1 to 2.4:1.Moreover, the Cistanche tubulosa extract is obtained from root materialand the Ginkgo biloba extract is obtained from leaf material. Inspecific embodiments, the Cistanche tubulosa extract is present in anamount of about 70 wt. % and the Ginkgo biloba extract is present in anamount of about 30 wt. %, each based on 100 parts by weight of thecomposition. It is contemplated that any and all values or ranges ofvalues between those described above may also be utilized.

In various embodiments, the Cistanche extract is present in an amount ofat least about 1, at least about 5, at least about 10, at least about15, at least about 20, at least about 25, at least about 50, at leastabout 100, at least about 150, at least about 200, at least about 250,at least about 300, at least about 350, at least about 400, at leastabout 450, or at least about 500, mg based on the total weight of thecomposition. In these embodiments, an upper boundary is generally≦5,000, ≦4,000, ≦3,000, ≦2,500, ≦2,000, ≦1,500, ≦1,000, ≦750, or ≦500,mg of Cistanche extract based on the total weight of the composition. Itis contemplated that any and all values or ranges of values betweenthose described above may also be utilized.

Surprisingly, it was discovered that a greater amount of Cistancheextract relative to Ginkgo extract provides synergistic benefits.Specifically, it was discovered that the extract combinations/mixturesdescribed herein provide beneficial effects on neuronal cell signaling.Moreover, it was discovered, unexpectedly, that an excess of Ginkgoextract relative to Cistanche extract (e.g. 50% or greater) can actuallyhave deleterious effects, specifically, by acting as an inhibitor ofdesired neuronal cell signaling. Other findings are described in theEXAMPLES section below.

The extracts can be obtained via conventional extraction methodsunderstood in the art, such as by water (e.g. steam) extraction or bysolvent (e.g. alcohol) extraction. The composition of this disclosure isnot limited to a particular extraction method, nor is extractionrequired since suitable extracts (e.g. standardized extracts) arereadily available from a number of commercial suppliers, such as fromSinphar Tian-Li Pharmaceutical, of Hongzhou, China and from BeijingGingko group of Beijing, China. Exemplarily extraction methods aredescribed below.

In order to obtain an extract, a polar solvent such as an alcohol (e.g.methanol, ethanol, butylene glycol), an ether (e.g. ethyl ether), aketone (e.g. acetone), an ester (e.g. ethyl acetate), water, or mixturesthereof, can be used as a solvent. Certain extracts can be also obtainedby further extracting the extract from the polar solvent with anon-polar solvent. Suitable non-polar solvents include, but are notlimited to, ethyl acetate, hexane, dichloromethane, chloroform, ormixtures thereof.

There are a variety of extraction methods that may be used to produceextracts suitable for the composition. These methods include, but arenot limited to, the extraction methods disclosed in U.S. Pat. No.7,897,184, which is hereby incorporated by reference in its entirety andpartially reproduced below with respect to some extraction methods.While extraction solvents described specifically mention ethanol, itshould be understood that other alcohols such as, but not limited to,isopropyl alcohol, ethyl alcohol, and/or methyl alcohol may be used inaddition to or as an alternative to ethanol. Exemplary alcoholicsolvents include, but are not limited to, C₁-C₄ alcohols, such asmethanol, ethanol, propanol, isopropanol, and butanol; hydro-alcohols ormixtures of alcohol and water, including hydro-ethanol; polyhydricalcohols, such as propylene glycol and butylene glycol; and fattyalcohols. Any of these alcoholic solvents may be used. Other solventssuch as, but not limited to, acetone may also be used as an extractionsolvent. Solvent-water blends, e.g. alcohol-water and/or acetone-waterblends, of any ratio, may also be used.

In one example, the extracts can be obtained using an organic solventextraction technique. In another example, solvent sequentialfractionation can be used to obtain the extracts. Total hydro-ethanolicextraction techniques can also be used to obtain the extracts.Generally, this is referred to as a lump-sum extraction. Extractsgenerated in the process will contain a broad variety of phytochemicalspresent in the extracted material including fat and water solublephytochemicals. Following collection of the extract solution, thesolvent will be evaporated, resulting in the extract.

Total ethanol extraction may also be used. This technique uses ethanolas the solvent. This extraction technique generates an extract that mayinclude fat soluble and/or lipophilic compounds in addition to watersoluble compounds. Total methanol extraction may also be used in asimilar manner with similar results.

Another example of an extraction technique that can be used to obtainthe extracts is supercritical carbon dioxide supercritical fluidextraction (“SFE”). In this extraction procedure, the material to beextracted is not exposed to any organic solvents. Rather, the extractionsolvent is carbon dioxide (CO₂), with or without a modifier, insuper-critical conditions (e.g. >31.3° C. and >73.8 bar). Those of skillin the art will appreciate that temperature and pressure conditions canbe varied to obtain the best yield of extract. This technique generatesan extract of fat soluble and/or lipophilic compounds, similar to totalhexane and ethyl acetate extraction techniques, which may also be used.

The composition can be prepared using various methods understood in theart. For example, actives of the composition, and optionally one or moreinactives, can be mixed or blended and compressed or compoundedutilizing various techniques understood in the art. The composition ofthis disclosure is not limited to a particular order of manufacturingsteps or method of manufacture.

Typically, the composition is administered (or ingested) orally, e.g.via the mouth (or “per os”). More typically, at least a portion of thecomposition is administered (or digested) enternally, e.g. via thegastrointestinal (GI) track (or “enteros”). The subject is typically ahuman, and can include men and women of various ages. Themethod/composition of this disclosure is not limited to a particularsubject.

The composition can be in various forms. Examples of suitable formsinclude solids, gels and liquids. Typically, the composition is solid.For example, the composition can be in the form of a pill, includingtablets, capsules, and caplets. In general, each of these terms can beused interchangeable in the art, e.g. tablet for pill or vice versa.Other than the Cistanche and Ginkgo extracts (i.e., the “actives” or“active ingredients”), the composition can include inactives (or“inactive ingredients”) including, but not limited to, excipients, suchas diluents and binders; granulating agents; glidants (or flow aids);fillers; lubricants; preservatives; stabilizers; coatings;disintegrants; sweeteners or flavors; and pigments. Further examples ofinactive ingredients are described above. In general, a number andquantity of excipients should be kept at a minimum as long as activeingredients are properly delivered. This is because subjects/consumerstend to prefer smaller tablets for easier consumption.

The composition can be in powder form, or pressed or compacted from apowder into a solid dose. A coating, e.g. polymer coating, may be usedto make the tablet smoother and easier to swallow, to control releaserate of the actives, to increase resiliency (or shelf life), and/or toenhance appearance. Other suitable oral forms of the composition includesyrups, elixirs, suspensions, and emulsions. Further non-limitingembodiments of the composition of this disclosure are describedhereafter.

In general, tablets provide a solid dosage form of delivery by oralroute. Typically, the main purpose of a tablet formulation is to deliveractive ingredients to a subject/consumer. Inactive ingredients areinactive substances that are generally used as carriers and formulationsupport for delivery of active ingredients. Inactive ingredients can beused for a variety of reasons, including handling small quantities (lowmg and mcg doses) of active ingredients, accurate dosing, stabilizingunstable active ingredients, degradation of active ingredients in thestomach, diluting active ingredients to prevent potential GI tractinjury, and/or masking unpleasant organoleptic properties (taste andsmell) of active ingredients.

Active ingredients must become biologically available to thesubject/consumer. For this purpose, active ingredients must first bedissolved and released into the body. For in vitro dissolution, the USPharmacopeia (“USP”) uses the following terms for determination of invitro dissolution profile of dosage forms: immediate release, extendedrelease and delayed release.

The in vivo release profile of active ingredients may be a conventional(unmodified) release, or a controlled release/sustained release (CR/SR),time release, targeted release or extended release. For CR/SR,zero-order kinetics is the ideal release profile. CR/SR profile maygenerally be achieved in two ways: (1) matrix, where active ingredientsare dispersed within a polymer (Example: 71G NF Carbopol® polymer,Lubrizol Advanced Material Inc.) or (2) reservoir, where active andinactive ingredients form the core, which is encapsulated bymembrane(s). Sometimes, a combination of different mechanisms is used(for example: CDT® controlled delivery technology, from SCOLR Pharma,which uses matrix erosion, changes in gel thickness, electrolyteionization, and ionic interactions mechanisms).

The in vivo release can also be enhanced for bioavailability. Severalfactors can influence the bioavailability of active ingredients,including release rate from the delivery system, active ingredientdegradation in the GI tract and poor permeability across gut mucosa.Some natural compounds have been shown to enhance the bioavailability ofa number of dietary ingredients in clinical trials, for example,BioPerine® from Sabinsa.

The composition can be administered in various amounts. In certainembodiments, the composition is administered in an amount to provide atleast about 1, at least about 5, at least about 10, at least about 15,at least about 20, at least about 25, at least about 50, at least about100, at least about 150, at least about 200, at least about 250, atleast about 300, at least about 350, at least about 400, at least about450, or at least about 500, mg of the Cistanche extract to the subject.In these embodiments, an upper boundary of administration is generally≦5,000, ≦4,000, ≦3,000, ≦2,500, ≦2,000, ≦1,500, ≦1,000, ≦750, or ≦500,mg of Cistanche extract to the subject. It is contemplated that any andall values or ranges of values between those described above may also beutilized.

The composition may be administered as needed, daily, several times perday or in any suitable regimen such that the desired outcome isachieved. In the method of this disclosure, the frequency ofadministration (e.g. of ingestion and/or digestion) can depend onseveral factors, including the desired level of memory improvement.Generally, a regimen includes administration of the composition once ortwice daily to include an administration in the morning and/or anadministration in the evening. The amount of composition administeredmay depend on several factors, including level of desired results andthe specific composition.

The composition can be used for improving memory of a subject. Invarious embodiments, the composition is administered to the subject on aperiodic basis, alternatively a daily basis, as part of a nutritionalsupplement regime for improving memory of the subject. In theseembodiments, one or more tablets of the composition, for example, may betaken per day. Typically, ingestion of the composition (e.g. in tabletform) coincides with a meal. Without being bound or limited by anyparticular theory, it is thought that ingestion of Ginkgo (biloba)provides quick support for mental performance, generally 4-6 hours aftertaking, while Cistanche (tubulosa) can improve memory, focus and/orrecall over a longer period of time, or a period of weeks, after taking.

The following examples, illustrating the compositions and methods ofthis disclosure, are intended to illustrate and not to limit theinvention. The Cistanche extract may simply be indicated as “C”, “CHE”,or “CT” in various examples or Figures. Moreover, the Ginkgo extract maysimply be indicated as “G” or “GB” in various examples or Figures.

EXAMPLES

A number of compositions representative of this disclosure areformulated and analyzed. Moreover, a number of comparative compounds areanalyzed and compared against the aforementioned compositions. Variousfindings/results and testing methodologies are generally illustrated inthe drawings, which are described in greater detail below.

Microelectrode array (MEA) neurochips were utilized to analyze variousexamples. Specifically, use of MEA neurochips enabled stimulation andrecording of bioelectricity with high spatial and temporal resolution,to glean neuroactivity of the Cistanche and Ginkgo extracts, alone andin combination, and with neuronal growth factors.

MEA neurochips are available from NeuroProof GmbH of Rostock, Germany.The electrophysiological properties of compounds can be evaluated usingtheir ability for induction of activity changes in neuronal networksgrown on MEA neurochips. Routine monitoring of internal dynamics ofmammalian neuronal networks is possible. The growth of neuronal networkson high-density MEA neurochips yields a hybrid test platform that allowsthe continuous and simultaneous monitoring of spike activity from alarge number of cells for weeks or even months. The advantage ofextracellular MEA-neurochip recording is the possibility of long-termrecording from multiple sites in vitro and the monitoring of signaltransmission between several hundred cells. Therefore, MEA neurochipsenable a real time analysis of action potential patterns at both thesingle cell and the whole network level while providing optical accessfor the observation of network architecture and growth.

Data from two major areas of experimentation show: 1) ACUTE applicationof Ginkgo and Cistanche extracts together at specific ratios exhibitsynergistic effects on neuronal cell signaling; and 2) CHRONIC treatmentof Cistanche altered neuronal responses to Neuron Growth Factor (“NGF”)over NGF alone.

Results: Acute Application

FIG. 1A depicts acute application examples, specifically bar charts froman isobolographic analysis of Ginkgo, Cistanche, and five mixturesthereof. It was found that a higher proportion of Ginkgo in the mixtureincreased the potency of the inhibitory action shown by effects at alower total concentration of the mixture (e.g. 20-50 μg/ml).

FIG. 1B further depicts acute application examples, specifically barcharts showing that a higher proportion of Cistanche in a mixtureresulted in a higher effect size. First activity enhancing effects wereobserved for mixtures containing 100%, 90%, and 75% Cistanche atconcentrations of 100 μg/ml. These effects were characterized by anincreased general activity, a strengthening in burst structure and astrengthening in the synchronicity.

FIG. 1C further depicts acute application examples, specifically barcharts showing that the enhancing effects increased with risingCistanche concentrations up to 250 μg/ml.

FIG. 1D further depicts acute application examples, specifically barcharts from the isobolographic analysis of Ginkgo (“G”), Cistanche(“C”), and the five mixtures thereof showing that the 25%/75% (G:C) andeven more so the 10%/90% (G:C) mixtures of Ginkgo/Cistanche appear tohave potentiating effects of the network simulation at concentrations of100 μg/ml, 150 μg/ml, and 250 μg/ml.

Results: Chronic Application

FIG. 2 depicts chronic application examples, specifically bar chartsthat show that the chronic treatment of hippocampal cultures with 300 nMDonepezil (“DPZ”) or 30 μg/ml Cistanche (“CHE”) during 4-28 days invitro enhanced the spontaneous network activity in both treatmentgroups. However, Cistanche induced stronger activity changes thanDonepezil.

FIG. 3 further depicts chronic application examples, specifically linegraphs that show that chronic treatment of hippocampal cultures with 300nM Donepezil or 30 μg/ml Cistanche during 4-28 days in vitro compensatedthe inhibitory acute NGF response. However, Cistanche induced a strongeracute compensation of NGF effects than Donepezil.

FIG. 4 further depicts chronic application examples, specifically barcharts that show that the chronic treatment of hippocampal cultures with300 nM Donepezil or 30 μg/ml Cistanche during 4-28 days in vitro inducedan increase in global synapse numbers. However, only Cistancherelatively increased the number of synapses per neurite.

FIG. 5A is an example image associated with semi-automaticquantification of neurons/image, neurites/image, synapses/image, andsynapse/neurite ratio. The image depicts color-merge of synapse (green),neurites (red), nuclei (blue), bar=50 μm.

FIG. 5B is an example image corresponding to synapse channel:color-inverted and analyzed for synapse particles (red labeled afterautomatic threshold, particle separation and size filter).

Methods

Research focused on the enhancement of neuronal network activitysuggestive of memory enhancement, through the evaluation of herbalextracts (i.e., Ginkgo and Cistanche). The method generally included thefour steps outlined below.

Step 1: Acute Study to Assess Effective Concentration Range

Primary mixed neuron/glia cultures from frontal cortex and hippocampusare plated onto MEA neurochips and cultured for at least four weeks.Acute application: effects of interaction between Cistanche and Ginkgoare analyzed for mixtures with different percentages of Cistanche(10-90%) and Ginkgo (the remainder) in addition to each alone.

FIG. 6 illustrates examples of the acute study to assess effectiveconcentration range. Seven different concentration response series(vertical) were performed, all with the same five concentrations(horizontal).

Step 2: Assess Combinatorial Effects of Ginkgo and Cistanche

FIG. 7A illustrates examples of the study to assess combinatorialeffects of Ginkgo and Cistanche, specifically a bar chart based on datapoints from all mixtures for one concentration at a time (native=100%).This Figure generally shows statistics versus “native”.

FIG. 7B further illustrates examples of the study to assesscombinatorial effects, specifically a line graph based on data pointsfrom all mixtures for one concentration and a hypothetical linear trendbetween Ginkgo and Cistanche (Native=0%). This Figure generally showsstatistics versus linear trend.

FIG. 7C further illustrates examples of the study to assesscombinatorial effects, specifically a line graph directed towarddetrending data, yielding the difference relative to the trend, andoverlaying all mixtures.

Step 3: Assess Chronic Effects of Cistanche tubulosa Extract onMaturation of a Neuronal Network

FIG. 8 illustrates examples of the study to assess chronic effects ofCistanche tubulosa extract on maturation of a neuronal network.Hippocampal cultures on MEA neurochips were treated with 30 μg/mlCistanche, 300 nM Donepezil, or a dimethyl sulfoxide (“DMSO”) vehiclecontrol. At 28 days after repeated dose chronic treatment, spontaneousactivity was measured, followed by recording of activity to acute NGFconcentration-response curve.

Step 4: Immuno-Histochemical Analysis

FIG. 9 illustrates examples of the study directed atimmuno-histochemical analysis. Recorded cultures are further analyzed byimmunocytochemistry, fluorescence microscopy and semi-automaticquantitative image analysis. Images are quantified per image for: cellnumber, neuronal number, neurite number, synapse number, % neurons,neurites per neuron, and synapses per neuron.

HPLC Fingerprinting

FIG. 10 illustrates a Cistanche tubulosa fingerprint. Specifically,standardization of Cistanche tubulosa using high-performance liquidchromatography (“HPLC”). The extract is standardized to 70%phenylethanoid glycosides (composed of at least 25% echinacoside,acetoside, isoacteoside, and verbacoside).

FIG. 11 illustrates a Ginkgo biloba fingerprint. Specifically,standardization of Ginkgo biloba using HPLC. The extract is standardizedto 24% flavone glycoside and 6% terpene lactones.

Phenotypic Screening with MEA Neurochips

MEA neurochip recordings were used to evaluate activity changes inhippocampal networks elicited by various concentrations and combinationsof two cultivated, standardized extracts of CT and GB, with and withoutacute application of NGF.

FIG. 12 illustrates steps of a phenotypic screening process utilizingMEA neurochips. The steps include: 1) neuronal cell culture; 2)phenotypic multichannel recording; 3) multi-parametric data analysis;and 4) pattern recognition. Each of these steps/aspects of FIG. 12 isdetailed below.

1) Primary murine cell culture: Frontal Cortex, Hippocampus, andHypothalamus.

2) Network spike trains and single neuron action potential. Highresolution of spatio-temporal network activity patterns.

3) 200 activity parameters: General Activity; Burst Structure;Synchronization/Connectivity; and Oscillation. Specific in vitro assayswith compound or disease related parameter selection.

4) Database with functional fingerprints of over 100 basic andclinically compounds. Similarity and differentiation from known compoundeffects. Combination effects.

FIG. 13 depicts neuron/glia co-cultures on MEA neurochips. The leftmostimage depicts GABAA receptor (alpha1, red), Neurons (TuJ, green), andnucei (blue); the second image from left depicts Neurons, neuronalsomata (HuCD, red), and nuclei; the second image from right depictsOligodendrocytes (04, red), Neurons, and nuclei; and the rightmost imagedepicts Astrocytes (GFAP, red), microglia (Lectin, green), and nuclei.

Characterization: Co-cultures of neurons and glial cells inserum-containing media e.g. cell populations in frontal cortex cultureson MEA neurochips (28 div): neurons (˜20%), astrocytes (˜70-80%),microglia (˜1-2%), oligodendrocytes (present, neglectable).

FIG. 14 illustrates multi-parametric characterization of neuronalnetwork activity.

Read out: Extracellular action potentials on single neuron and networkactivity level; spatio-temporal network activity changes in time scalesof spikes and bursts. Spike train is described by 200 activityparameters in four categories: 1. General Activity, e.g. spike rate,burst rate, burst period, and percent of spikes in burst; 2. BurstStructure, e.g. number, frequency and ISI of spikes in bursts, burstduration, amplitude, area, plateau position, and plateau duration; 3.Oscillation, e.g. variation over time as an indicator for the strengthof the oscillation; Gabor function fitted to autocorrelograms; and 4.Synchronicity/Connectivity, e.g. variation within the network as anindicator for the strength of the synchronization, simplexsynchronization, and percent of units in synchronized burst.

Acute Application of Ginkgo biloba and Cistanche tubulosa

Comparison of the acute concentration-dependent effects induced by A) GBand B) CT and their respective DMSO control concentrations onhippocampus network activity in vitro. Displayed are six activitydescribing parameters in four categories (general activity, burststructure, oscillation, and synchronization) for treatment of nineconcentrations from 10 pg/ml to 100 μg/ml. (mean±standard error, Ginkgo:n=5, Cistanche: n=8, DMSO: n=5. Student's unpaired t-test: *p≦0.05;**p≦0.01; ***p≦0.001).

FIG. 15 depicts line graphs illustrating that acute application ofGinkgo biloba induces a mild concentration dependent activity change inburst structure. FIG. 16 depicts line graphs illustrating that acuteapplication of Cistanche tubulosa induces a mild concentration dependentbi-phasic activity change in burst structure.

Isobolographic Analysis

FIG. 17 depicts Ginkgo and Cistanche mixture and concentration matricesassociated with general activity (“GA”), burst structure (“BS”),oscillation (“OS”), and synchronicity (“SY”).

To make the matrices, data from concentration response curves are colorcoded as increased or decreased relative to native. Next, results fromall mixtures and all concentrations are combined into a matrix. Next,out of 200, the relevant activity features describing GA, BS, OS, and SYare compared to a reference fingerprint.

Higher % of GB induces changes at lower concentrations of totalcompound. Increasing amounts of GB (>50%) elicited an inhibition ofgeneral activity and when GB:CT is 75%/25%, surprisingly the inhibitionpotency increased five-fold over GB alone. Mixtures with increasing CTpotentiated network stimulation, most apparent at 10%/90% GB:CT and 100μg/ml, 150 μg/ml, and 250 μg/ml.

Chronic Effects

FIG. 18A depicts bar charts illustrating chronic effects. Specifically,chronic effects on native activity of 30 μg/ml Cistanche tubulosa(“CT”), two CT and Ginkgo biloba (“GB”) mixtures in a ratio of 90%:10%or 70%:30% (each at 10 μg/ml or 30 μg/ml), or 0.021% DMSO vehiclecontrol.

FIG. 18B depicts additional bar charts illustrating the chronic effects.In all of the bar charts, the leftmost bar is 0.021% DMSO, second barfrom left is 10 μg/ml at GB10+CT90, third bar from left is 30 μg/ml atGB10+C90, third bar from right is 10 μg/ml at GB30+CT70, second bar fromright is 30 μg/ml at GB30+CT70, and rightmost bar is 30 μg/ml at 100%CT. (mean±standard error, student's t-test: *p≦0.05; **p≦0.01;***p≦0.001).

CT alone resulted in an enhancement of spontaneous activity throughstrengthened bursting activity, particularly increase of burst surpriseand increase of percentage of total spikes grouped in bursts. Chronictreatment of 10 μg/ml of the 70:30 mixture (CT:GB) caused networkactivity changes, notably an increase in spike rate, decrease in eventrate and loosening of burst structure.

Heat Mapping

FIG. 19A is a first portion of a heat map and FIG. 19B is a secondportion of the heat map. The heat map illustrates concentrationdependent effects of acute mouse nerve growth factor (“mNGF”) on networkactivity of chronically treated hippocampal cultures with the mixturesor components described above for FIG. 18.

The heat map illustrates significant changes on the 60 mostrepresentative parameters for each mNGF concentration, from 3 ng/ml to 1μg/ml. The color code changes in activity parameters according to thepercent changes (100:no change), with yellow/red=an increase andgreen/blue=a decrease.

NGF alone induced an inhibition of general activity. Chronic CT alonecompensated for the inhibition seen after NGF. The mixture of GB30:CT70at both concentrations evoked the opposite effect from CT alone, as itenhanced the acute mNGF effect.

Effect of Chronic Application

FIG. 20A is a bar chart illustrating chronic application effects onhippocampal network morphology with respect to total number ofcells/field. FIG. 20B is a bar chart illustrating chronic applicationeffects on hippocampal network morphology with respect to percentage ofneurons. FIG. 20C is a bar chart illustrating chronic applicationeffects on hippocampal network morphology with respect to neuriticdensity. FIG. 20D is a bar chart illustrating chronic applicationeffects on hippocampal network morphology with respect to synapticnumber/field. The mixtures or components for FIG. 20 are as describedabove for FIG. 18.

Recorded cultures were further analyzed by immunocytochemistry,fluorescent microscopy and quantitative image analysis for morphologicalfindings. Semi-automatic quantification (mean±SEM, student's t-test:*p≦0.05; n=3 with 10-20 images each.)

FIG. 21 are images associated with semi-automatic quantification oftotal cells/field, percentage of neurons, neuritic density, and synapticnumber/field. The leftmost images correspond to color-merge of synapse(green), neuritis (red), and nuclei (blue). The rightmost imagescorrespond to synapse channel: color inverted and analyzed for synapseparticles. After analysis, a higher number or synapses are qualitativelyobserved in CT 30 μg/ml treated cultures.

CT alone did not increase neuronal cell number, but did increase thenumber of global synapses as well as the number of synapses per neurite.CT70:GB30 at 30 μg/ml also induced morphological effects shown by anincreased percentage of neurons (+27%), and increased number of synapsesin relation to the neuritic density increased by 31%.

Summary of Non-Limiting Theories, Examples/Analysis, and Conclusions

Prior to this disclosure, effects of Cistanche and Ginkgo extracts onneural activity were not clearly elucidated. As described above, MEAneurochip recordings were used to evaluate activity changes inhippocampal networks elicited by various concentrations and combinationsof two cultivated, standardized extracts of CT and GB. After acuteapplication to four week old primary hippocampus cultures,multi-parametric analysis revealed both CT and GB induced mild butmeasurable activity changes within four functional activity categories:GA, BS, OS, and SY.

The isobolographic approach revealed interaction between GB and CT atspecific concentrations becoming most apparent at 100 μg/ml when theGB/CT combination is composed of 10-25% GB (remainder CT), with theeffects of 10% GB being most pronounced. This concentration andcombination increased the spike organization into bursts, induced astronger BS and increased bursting regularity and SY. Increasing amountsof GB (>50%) elicited an inhibition of GA. Further, when GB:CT is 75/25%potency of GB increased five-fold over GB alone. Mixtures withincreasing CT potentiated network stimulation, most apparent at 10%/90%(GB:CT) and 100 μg/ml, 150 μg/ml, and 250 μg/ml.

Chronic CT treatment (30 μl/ml) of hippocampal cultures on MEAneurochips from 4-28 days in vitro resulted in an enhancement ofspontaneous activity through strengthened bursting activity. NGF appliedacutely to CT treated cultures further increased the response over thatof NGF applied to vehicle-treated cultures, notably increasing GA,lengthening burst duration, increasing pattern regularity, and improvingSY within the networks.

Recorded cultures were further analyzed by immunocytochemistry,fluorescent microscopy and quantitative image analysis for morphologicalfindings. Chronically treating hippocampal networks with CT did notincrease neuronal cell number, but did increase the number of globalsynapses as well as the number of synapses per neurite. These findingssupport the notion that CT and GB are neuro-active and interact withendogenous growth factors within neural systems. Further, it has beenshown that chronic repeated-dose treatment with CT induced morphologicalalterations and increased hippocampal network activity in vitro.

Synergistic Effects of Ginkgo and Cistanche Extracts on NeuronalActivation

The examples above provide scientific evidence to support synergisticinteraction between Ginkgo and Cistanche on neuronal network activity invitro, and scientifically support the positioning of Ginkgo andCistanche combination in humans. Further, the examples above illustratea mechanism of action for memory improvement for Cistanche.

A surprising and profound discovery was that Ginkgo supports the actionof Cistanche in a synergistic way and that chronic treatment withCistanche in vitro induced an increase in the global number of synapsesand the number of synapses per neurite. This allows for spatial andtemporal consolidation of neuronal signal which is known to play a keyrole in long term potentiation (LTP), the neural correlate of memory.

The results found were equal or better than the results from thepositive control pharmaceutical compound (i.e., Donepezil; trade nameAricept) which is widely marketed for enhancement of cognition as anacetlycholinesterase (AChE or acetylhydrolase) inhibitor.

Neuronal Data Gathering and Analysis

Multichannel recording delivered single neuron spike data and spikeidentification and separation were accomplished with a template-matchingalgorithm in real time, to allow the extracellular recording of actionpotentials from 256 neurons simultaneously. The action potentials or“spikes” were recorded in spike trains and clustered in bursts, whichcan be quantitatively described via direct spike train analysis. Highcontent analysis of the network activity patterns provides amulti-parametric description characterizing the activity in fourcategories: GA, BS, SY and OS. From the spike trains generated by thisanalysis, a total of 200 activity-describing spike train parameters weredetermined for each of these four categories.

Predominate Findings Using these Methods—Acute Application to CellCultures

Both Cistanche and Ginkgo extracts applied acutely to neuronal culturesaffects network activity, Cistanche more so than Ginkgo with Cistanchechanging GA and BS but Ginkgo only changing BS mildly. Further, asimilarity was observed between the actions of a pharmaceutical drugmarketed for cognitive improvement (i.e., Donepezil) and Cistanche interms of the direction of parameter shifts, but this parameter shiftingwas not seen after acute application of Ginkgo.

Activity enhancing effects were observed for mixtures of Cistanche andGinkgo as follows: 10% and 25% Ginkgo (remainder Cistanche) made neuronsmore sensitive to the actions of Cistanche in that GA, strengthening ofthe BS and stronger SY was induced at a lower overall concentration.Further, at this dilution (10-25% Ginkgo and 90-75% Cistanche) networkstimulation was potentiated with a higher effect size.

Predominate Findings Using these Methods—Chronic Application to CellCultures

24 days of Cistanche treatment of hippocampal cell cultures in vitroenhanced spontaneous network activity, similar to that seen with chronicapplication of Donepezil, except that Cistanche effects were morepronounced than those of the pharmaceutical. Both Donepezil andCistanche Induced an increase in global synapse numbers, but onlyCistanche increased the number of synapses per neurite

By using MEA neurochips to capture the neuronal activation patterns ofthe Cistanche and Ginkgo extracts on hippocampal cell cultures,synergistic activity has been shown when the Cistanche extract isdiluted to 75-90% with 25-10% of Ginkgo extract. It has also been shownthat chronic treatment of Cistanche can induce neuronal morphologychanges that are indicative of improved synaptic connectivity to supportlong term potentiation and memory.

Ratios of Ginkgo and Cistanche have been identified that producesynergistic effects on neuronal activity patterns, including makingneurons more sensitive to the actions of Cistanche; GA, strengthening ofthe BS and stronger SY between the firing of individual neurons.Further, at various dilutions, the combination of Cistanche and Ginkgopotentiated network stimulation with a higher effect size. In general ahigh level of Ginkgo (>50%) in combination with Cistanche will reducethe synergistic effect of the combination.

Moreover, the examples of this disclosure show that Cistanche can workalongside the natural neuronal strengthening effects of endogenous NGF.NGF is known to augment neuronal survival and to modify synapticefficacy and neuronal plasticity. Staying mentally active, pursuingactivities that bring mental satisfaction, and staying active throughexercise, are positive behaviors for mental health. Each of thesebehaviors also increases endogenous production of NGFs in the brain.Thus, the combination of such behaviors and the compositions of thisdisclosure make a natural pairing with one another as complementary waysto strengthen and protect the longevity and robustness of cognitiveabilities as a subject ages.

The terms “comprising” or “comprise” are used herein in their broadestsense to mean and encompass the notions of “including”, “include”,“consist(ing) essentially of”, and “consist(ing) of”. The use of “forexample”, “e.g.”, “such as”, and “including” to list illustrativeexamples does not limit to only the listed examples. Thus, “for example”or “such as” means “for example, but not limited to” or “such as, butnot limited to” and encompasses other similar or equivalent examples.The term “about” as used herein serves to reasonably encompass ordescribe minor variations in numerical values measured by instrumentalanalysis or as a result of sample handling. Such minor variations may bein the order of ±0-10, ±0-5, or ±0-2.5, % of the numerical values.Further, The term “about” applies to both numerical values whenassociated with a range of values. Moreover, the term “about” may applyto numerical values even when not explicitly stated.

Generally, as used herein a hyphen “-” or dash “—” in a range of valuesis “to” or “through”; a “>” is “above” or “greater-than”; a “≧” is “atleast” or “greater-than or equal to”; a “<” is “below” or “less-than”;and a “≦” is “at most” or “less-than or equal to”. On an individualbasis, each of the aforementioned applications for patent, patents,and/or patent application publications, is expressly incorporated hereinby reference in its entirety in one or more non-limiting embodiments.

It is to be understood that the appended claims are not limited toexpress and particular compounds, compositions, or methods described inthe detailed description, which may vary between particular embodimentswhich fall within the scope of the appended claims. With respect to anyMarkush groups relied upon herein for describing particular features oraspects of various embodiments, it is to be appreciated that different,special, and/or unexpected results may be obtained from each member ofthe respective Markush group independent from all other Markush members.Each member of a Markush group may be relied upon individually and or incombination and provides adequate support for specific embodimentswithin the scope of the appended claims.

It is also to be understood that any ranges and subranges relied upon indescribing various embodiments of the present invention independentlyand collectively fall within the scope of the appended claims, and areunderstood to describe and contemplate all ranges including whole and/orfractional values therein, even if such values are not expressly writtenherein. One of skill in the art readily recognizes that the enumeratedranges and subranges sufficiently describe and enable variousembodiments of the present invention, and such ranges and subranges maybe further delineated into relevant halves, thirds, quarters, fifths,and so on. As just one example, a range “of from 0.1 to 0.9” may befurther delineated into a lower third, i.e., from 0.1 to 0.3, a middlethird, i.e., from 0.4 to 0.6, and an upper third, i.e., from 0.7 to 0.9,which individually and collectively are within the scope of the appendedclaims, and may be relied upon individually and/or collectively andprovide adequate support for specific embodiments within the scope ofthe appended claims. In addition, with respect to the language whichdefines or modifies a range, such as “at least,” “greater than,” “lessthan,” “no more than,” and the like, it is to be understood that suchlanguage includes subranges and/or an upper or lower limit. As anotherexample, a range of “at least 10” inherently includes a subrange of fromat least 10 to 35, a subrange of from at least 10 to 25, a subrange offrom 25 to 35, and so on, and each subrange may be relied uponindividually and/or collectively and provides adequate support forspecific embodiments within the scope of the appended claims. Finally,an individual number within a disclosed range may be relied upon andprovides adequate support for specific embodiments within the scope ofthe appended claims. For example, a range “of from 1 to 9” includesvarious individual integers, such as 3, as well as individual numbersincluding a decimal point (or fraction), such as 4.1, which may berelied upon and provide adequate support for specific embodiments withinthe scope of the appended claims.

The present invention has been described herein in an illustrativemanner, and it is to be understood that the terminology which has beenused is intended to be in the nature of words of description rather thanof limitation. Many modifications and variations of the presentinvention are possible in light of the above teachings. The presentinvention may be practiced otherwise than as specifically describedwithin the scope of the appended claims. The subject matter of allcombinations of independent and dependent claims, both single andmultiple dependent, is herein expressly contemplated.

What is claimed is:
 1. A method for improving memory of a subject, saidmethod comprising the step of: administering a composition to thesubject; wherein the composition consists essentially of Cistancheextract and Ginkgo extract in a weight ratio (C:G) that is >2.5:1 or<2.5:1.
 2. The method as set forth in claim 1, wherein the weight ratiois from 2.6:1 to 20:1, alternatively 3:1 to 9:1.
 3. The method as setforth in claim 2, wherein: i) the Cistanche extract is present in anamount of from about 72-99 wt. %, alternatively about 75-90 wt. %, basedon 100 parts by weight of the composition; ii) the Ginkgo extract ispresent in an amount of from about 1-28 wt. %, alternatively about 10-25wt. %, based on 100 parts by weight of the composition; or iii) both i)and ii).
 4. The method as set forth in claim 3, wherein: i) theCistanche extract is further defined as Cistanche tubulosa extract; ii)the Cistanche extract is obtained from root material of a plant in thegenus Cistanche; or iii) both i) and ii).
 5. The method as set forth inclaim 4, wherein: i) the Ginkgo extract is further defined as Ginkgobiloba extract; ii) the Ginkgo extract is obtained from leaf material ofa plant in the genus Ginkgo; or iii) both i) and ii).
 6. The method asset forth in claim 5, wherein: i) the composition is administeredorally; ii) the composition is in the form of a tablet; or iii) both i)and ii).
 7. The method as set forth in claim 1, wherein: i) theCistanche extract is present in an amount of from about 72-99 wt. %,alternatively about 75-90 wt. %, based on 100 parts by weight of thecomposition; ii) the Ginkgo extract is present in an amount of fromabout 1-28 wt. %, alternatively about 10-25 wt. %, based on 100 parts byweight of the composition; or iii) both i) and ii).
 8. The method as setforth in claim 1, wherein the weight ratio is from 1:1 to 2.4:1,alternatively 3:2 to 7:3.
 9. The method as set forth in claim 8,wherein: i) the Cistanche extract is present in an amount of from about50-70 wt. %, alternatively about 60-70 wt. %, based on 100 parts byweight of the composition; ii) the Ginkgo extract is present in anamount of from about 30-50 wt. %, alternatively about 30-40 wt. %, basedon 100 parts by weight of the composition; or iii) both i) and ii). 10.The method as set forth in claim 9, wherein: i) the Cistanche extract isfurther defined as Cistanche tubulosa extract; ii) the Cistanche extractis obtained from root material of a plant in the genus Cistanche; oriii) both i) and ii).
 11. The method as set forth in claim 10, wherein:i) the Ginkgo extract is further defined as Ginkgo biloba extract; ii)the Ginkgo extract is obtained from leaf material of a plant in thegenus Ginkgo; or iii) both i) and ii).
 12. The method as set forth inclaim 11, wherein: i) the composition is administered orally; ii) thecomposition is in the form of a tablet; or iii) both i) and ii).
 13. Themethod as set forth in claim 1, wherein: i) the Cistanche extract ispresent in an amount of from about 50-70 wt. %, alternatively about60-70 wt. %, based on 100 parts by weight of the composition; ii) theGinkgo extract is present in an amount of from about 30-50 wt. %,alternatively about 30-40 wt. %, based on 100 parts by weight of thecomposition; or iii) both i) and ii).
 14. The method as set forth inclaim 1, wherein the composition is administered in an amount to provideat least about 250 mg, alternatively at least about 300 mg, of theCistanche extract to the subject.
 15. The method as set forth in claim1, wherein the composition is administered to the subject on a periodicbasis, alternatively a daily basis, as part of a nutritional supplementregime for improving memory of the subject.
 16. A composition foradministration to a subject for improving memory of the subject, saidcomposition consisting essentially of Cistanche extract and Ginkgoextract in a weight ratio (C:G) that is >2.5:1 or <2.5:1.
 17. Thecomposition as set forth in claim 16, wherein the weight ratio is from2.6:1 to 20:1, alternatively 3:1 to 10:1.
 18. The composition as setforth in claim 17, wherein: i) the Cistanche extract is present in anamount of from about 72-99 wt. %, alternatively about 75-90 wt. %, basedon 100 parts by weight of the composition; ii) the Ginkgo extract ispresent in an amount of from about 1-28 wt. %, alternatively about 10-25wt. %, based on 100 parts by weight of the composition; or iii) both i)and ii).
 19. The composition as set forth in claim 16, wherein theweight ratio is from 1:1 to 2.4:1, alternatively 3:2 to 7:3.
 20. Thecomposition as set forth in claim 19, wherein: i) the Cistanche extractis present in an amount of from about 50-70 wt. %, alternatively about60-70 wt. %, based on 100 parts by weight of the composition; ii) theGinkgo extract is present in an amount of from about 30-50 wt. %,alternatively about 30-40 wt. %, based on 100 parts by weight of thecomposition; or iii) both i) and ii).
 21. The composition as set forthin claim 16, wherein: i) the Cistanche extract is further defined asCistanche tubulosa extract; ii) the Cistanche extract is obtained fromroot material of a plant in the genus Cistanche; or iii) both i) andii).
 22. The composition as set forth in claim 16, wherein: i) theGinkgo extract is further defined as Ginkgo biloba extract; ii) theGinkgo extract is obtained from leaf material of a plant in the genusGinkgo; or iii) both i) and ii).
 23. The composition as set forth inclaim 16, comprising: about 72-99 wt. % Cistanche tubulosa extract; andabout 1-28 wt. % Ginkgo biloba extract; wherein the Cistanche tubulosaand Ginkgo biloba extracts are present in a weight ratio (C:G) that isfrom 2.6:1 to 20:1, the Cistanche tubulosa extract is obtained from rootmaterial, and the Ginkgo biloba extract is obtained from leaf material.24. The composition as set forth in claim 23, wherein the Cistanchetubulosa extract is present in an amount of about 90 wt. % and theGinkgo biloba extract is present in an amount of about 10 wt. %, eachbased on 100 parts by weight of the composition.
 25. The composition asset forth in claim 23, wherein the Cistanche tubulosa extract is presentin an amount of about 75 wt. % and the Ginkgo biloba extract is presentin an amount of about 25 wt. %, each based on 100 parts by weight of thecomposition.
 26. The composition as set forth in claim 16, comprising:about 50-70 wt. % Cistanche tubulosa extract; and about 30-50 wt. %Ginkgo biloba extract; wherein the Cistanche tubulosa and Ginkgo bilobaextracts are present in a weight ratio (C:G) that is from 1:1 to 2.4:1,the Cistanche tubulosa extract is obtained from root material, and theGinkgo biloba extract is obtained from leaf material.
 27. Thecomposition as set forth in claim 26, wherein the Cistanche tubulosaextract is present in an amount of about 70 wt. % and the Ginkgo bilobaextract is present in an amount of about 30 wt. %, each based on 100parts by weight of the composition.
 28. The composition as set forth inclaim 16, wherein: i) the Cistanche extract comprises at least onephenylethanoid glycoside; ii) the Ginkgo extract comprises at least oneflavonoid and at least one terpenoid; or iii) both i) and ii).